ES2554835T3 - Cell line identification procedure - Google Patents

Abstract

A cell line identification procedure comprising the following steps: (a) calmodulin gene analysis, in which a fragment of the calmodulin gene is amplified by PCR and in which the amplified fragment comprises the sequence between exon 3 and exon 4 of the calmodulin gene; and (b) analysis of the Axl receptor tyrosine kinase gene, in which a fragment of the Axl receptor tyrosine kinase gene is amplified by PCR, and in which the amplified fragment comprises the sequence between exon 18 and exon 19 of the Axl receptor tyrosine kinase gene; and in which the cell line is identified by the size of an intron in said genes.

Description

Cell line identification procedure

Field of the Invention

The present invention relates to the field of cell line identification. More particularly, it refers to the identification of cell lines by analysis by molecular techniques, and the identification of variation in introns or determining whether a particular gene is present. This procedure is particularly useful for the quality control of the cell lines used for the production of pharmaceutical preparations.

Background of the invention

Current procedures performed in quality control (QC) use the isoenzyme technique. This procedure is based on the analysis of electrophoretic mobility of isoenzymes that differ between species. Mobility profiles can be established for each species using the combined analysis of the migration distances of the enzymes, such as: glucose-6-phosphate dehydrogenase (G6PD), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and malate dehydrogenase (MD). This procedure provides a means to determine the species of origin of a cell line and detect cells with transverse contamination with cells of another cell line different species (Stacey and Hockley, 2006). However, this test is laborious and time consuming. Liu et al. (2008) disclose the identification and authentication of closely related animal cell culture by PCR amplification of the aldolase gene.

Summary of the invention

The present inventors have shown that cell lines can be identified by a more reliable and easier procedure than with current identification methodologies. The identification procedure of the present invention is based on the analysis of the size of the intronic sequences that differ between species. This procedure involves the amplification of the intronic sequences by the polymerase chain reaction (PCR). The present inventors have identified genes that are sufficiently conserved in the exonic sequences to allow hybridization with the PCR amplification primers and sufficiently different in the intron sequence to allow discrimination based on length polymorphism. Therefore, the discrimination of these variations can be determined by analysis of these genomic regions.

The procedure allows the identification of a series of vertebrate-derived cell line types (for example, MRC-5, Vero, CHO, FRHL-2, MDCK, chicken embryo fibroblasts [CEF]), based on informative polymorphic introns that They are found in two genes, calmodulin and Axl receptor tyrosine kinase. In addition, using a specific insect cell line gene, analysis to determine the presence or absence of an insect gene can help identify insect-derived cell lines such as Hi-5.

Accordingly, the present invention provides a cell line identification method comprising one or more of the following steps:

(to)

calmodulin gene analysis, in which a fragment of the calmodulin gene is amplified by PCR and in which the amplified fragment comprises the sequence between exon 3 and exon 4 of the calmodulin gene; Y

(b)

(c) analysis of the Axl receptor tyrosine kinase, in which a fragment of the Axl receptor tyrosine kinase gene is amplified by PCR, and in which the amplified fragment comprises the sequence between exon 18 and exon 19 of the gene of Axl receptor tyrosine kinase;

and in which the cell line is identified by the size of an intron in said genes.

Brief description of the figures

Figure 1 Analysis of the CALM3 / 4 PCR products

Figure 2 Analysis of the AXL 18/19 PCR products.

Figure 3 Analysis of the PPATTA PCR product.

Description of the invention

The inventors claim that the terms "comprising", "comprises" and "consisting", may optionally be substituted with the expressions "consisting of", "consisting of" "constituted by", respectively, in each case.

The present inventors have identified conserved regions and variables in the genes coding for calmodulin and receptor tyrosine kinase Axl. Variation in genes allows discrimination between different cell lines; These variations can be detected using any procedure that allows nucleic acid analysis. Additionally, conserved regions can be used to design complementary primers, either for PCR amplification or for sequencing reaction that can be used.

to analyze cell lines from many sources. Insect-derived cell lines can be identified by detecting insect-specific nucleotide sequences.

In accordance with the foregoing, the present invention provides a method of identifying the cell line as defined in the claims.

The present invention can be used to identify many types of cell lines. The term "cell line" as used herein refers to established cell cultures grown in vitro. Cell lines can be derived from any species of animals, including mammals, birds and insects. In a particular embodiment, the identification method of the present invention is used for cell lines used in the pharmaceutical industry. In a particular embodiment, the methods of the present invention are used to identify cell lines used in the production of viruses that can be used in vaccines. The term "cell line" encompasses continuous cell cultures that are cell cultures of morphologically uniform cells. which can be propagated in vitro for an indefinite time. According to the above, in one embodiment, the methods of the invention can be used to identify and / or differentiate the selected cell lines from the group: MRC-5, Vero, Hi-5, CHO, FRHL2 and MDCK. The term "cell line" also incorporates primary cell lines, that is, cells that are isolated directly from a subject. In accordance with the foregoing, in one embodiment, the methods of the present invention are used to identify chicken-derived cells, for example, CEF (chicken embryo fibroblasts).

The present inventors have identified calmodulin and receptor tyrosine kinase Axl genes, which comprise conserved exons and variable introns. In accordance with the foregoing, in one aspect of the disclosure, there is provided a method of identifying the cell line comprising the step of: analysis of the calmodulin gene. In a further aspect, a method of identifying the cell line is provided which comprises the step of: analysis of the Axl receptor tyrosine kinase gene.

A specific insect gene has also been identified, which allows discrimination between cells derived from insects and cells derived from other organisms, for example mammalian or bird cells. In accordance with the foregoing, in a further aspect of the disclosure, there is provided a method of identifying the cell line comprising the step of: analysis of an atacin gene.

The term "analysis" refers to any analytical technique that will allow the skilled person to differentiate / discriminate nucleic acid sequences from different cell lines. There are a large number of techniques suitable for this purpose known to those skilled in the art, these techniques include, but are not limited to: polymerase chain reaction amplification (PCR), sequencing, hybridization and restriction fragment length polymorphisms (RFLP ). In one aspect of the disclosure, a cell line identification method of the invention is provided in which the analysis procedure is selected from the group consisting of polymerase chain reaction (PCR) amplification, sequencing, hybridization and polymorphisms. Restriction Fragment Length (RFLP).

The term "calmodulin gene" is well known to the person skilled in the art and as used herein it is understood to mean a gene that encodes a polypeptide comprising at least one EFmano motif that is capable of binding to the calcium. Calmodulin plays an important role in the transmission of intracellular Ca2 + signals to a wide variety of effector systems (Rhyner et al., (1994) Euro J Biochem 225: 71-81). In mammals, 3 genes (CALM1, CALM2 and CALM3) encode the highly conserved individual protein, calmodulin (CaM) (Berchtold et al., (1993) Genomics 16: 461-465). In particular embodiments of the invention, the term "calmodulin gene" is a nucleic acid sequence that encodes a polypeptide of SEQ ID No. 7 or encodes an amino acid sequence that has a sequence identity of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO. 7.

The term "atacin gene" as used herein refers to nucleic acid sequences encoding antibacterial peptides of insects known as atacins and atacin-like proteins including basic and acidic atacins (see, for example, Boman et al. ., (1991) Eur J. Biochem 201: 21-31). The term encompasses genomic sequences encoding atacins, proatacins and pre-proatacins; These terms are well known to those skilled in the art. Atacins are polypeptides that are specific to insects and, therefore, are not found in mammals or birds. In specific aspects of the disclosure, the expression "atacin gene" is a nucleic acid sequence encoding a polypeptide of SEQ ID NO. 9 or polypeptides with amino acid sequences having a sequence identity of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO. 9.

The term "Axl receptor tyrosine kinase" is well known to one skilled in the art and is used herein to mean transmembrane proteins that participate in intracellular signaling that is triggered through the activation of the tyrosine kinase domain and the subsequent phosphorylation of various substrates and that is a part of the tyrosine kinase receptor of the Axl family. Axl receptor tyrosine kinases are ubiquitously expressed in a series of cell lines, including those of epithelial, mesenchymal and hematopoietic origin, as well as non-transformed cells (Hafizi and Dahlhbäck, 2006 [Cytokine & Growth Factor Reviews 17: 295-304 ]).

The transforming activity of the receptor tyrosine kinase Axl demonstrates that the receptor can lead to cell proliferation. Although the role of Axl receptor tyrosine kinase in cells and non-transformed tissues was unknown, Varnum et al. (1995, Nature, 373: 623-626) suspected that it may involve stimulation of cell proliferation in response to a suitable signal, that is, a ligand that activates the receptor. Varnum et al. (1995) purified the Axl receptor tyrosine kinase stimulating factor and identified as the product of growth-specific gene 6 (GAS6). The Axl tyrosine kinase gene encodes a tyrosine kinase receptor that regulates cell growth and differentiation (O'Bryan et al., (1991) Mol Cell Biol 11: 5016-31). The genomic organization of the human Axl tyrosine kinase gene locus was described by Schulz et al., (1993 Oncogene 8: 509-13). The human Axl tyrosine kinase gene contains 20 exons that are distributed over a 44 kb region.

In particular embodiments of the invention, the expression "receptor tyrosine kinase gene AxI" means a nucleic acid sequence encoding a polypeptide of SEQ ID NO: 8 or polypeptides with amino acid sequences having a sequence identity of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO. 8.

In a further aspect, a cell line identification procedure is provided comprising the following steps: (a) calmodulin gene analysis; and (b) analysis of the Axl receptor tyrosine kinase gene.

In a further aspect, a cell line identification procedure is provided comprising the following steps: (a) calmodulin gene analysis; and (b) analysis of an atacin gene.

In one aspect, a cell line identification procedure is provided comprising the following steps: (a) analysis of an atacin gene; and (b) analysis of the Axl receptor tyrosine kinase gene.

In a further aspect, a cell line identification procedure is provided comprising the following steps: (a) calmodulin gene analysis; (b) analysis of the Axl receptor tyrosine kinase gene; Y

(c) analysis of an attack gene.

It is envisioned that the expert may wish to use various molecular techniques to identify one or more cell lines. For example, in certain aspects of the disclosure the expert can use one or more, or any combination of the following techniques: PCR, sequencing, hybridization and RFLP. In accordance with the foregoing, the methods of the disclosure may be carried out using one or more of the molecular techniques selected from the list comprising PCR, sequencing, hybridization and RFLP, or any combination thereof.

In a particular aspect of the disclosure, there is provided a method of identifying the cell line comprising the steps comprising one or more of the following steps: (a) PCR amplification of a calmodulin gene or fragment thereof; (b) PCR amplification of an Axl receptor tyrosine kinase gene or fragment thereof; or (c) PCR amplification of an atacin gene or fragment thereof; or any combination thereof.

The term "fragment" as described herein refers to any nucleic acid sequence of a gene, which is not the entire gene sequence, comprising at least 50, 60, 70, 80, 90, 100, 110 , 120, 130, 140, 150, 160, 170, 180, 190, 200, 400, 600, 800, 1,000, 1,200, 1,400, 1,600 or more nucleotides.

In a further aspect of the disclosure, a cell line identification procedure is provided comprising one or more of the following steps: (a) hybridization of a probe to the calmodulin gene or fragment thereof; (b) hybridization of a probe to an Axl receptor tyrosine kinase gene or fragment thereof; or (c) hybridization of a probe to an atacin gene or fragment thereof; or any combination thereof.

In one aspect of the disclosure, a cell line identification procedure is provided comprising one or more of the following steps: (a) sequencing of the calmodulin gene or fragment thereof;

(b) sequencing of an Axl receptor tyrosine kinase gene or fragment thereof; or (c) sequencing of an atacin gene or fragment thereof; or any combination thereof.

Genes encoding calmodulin comprise introns of varying size between different cell lines.

According to the above, in one aspect, the cell line is identified by the size (number of nucleotides) of the amplicon (product amplified after PCR). The inventors have determined that introns within the receptor tyrosine kinase Axl and calmodulin genes vary in size between different species and / or strains. In accordance with the foregoing, in a particular aspect of the disclosure cell lines can be differentiated and / or determined by the size of a particular intron between two exons. In a particular aspect, cell lines are determined by the size of the amplicon derived from the calmodulin gene and / or the receptor tyrosine kinase gene Axl. In one aspect, a method of identifying the cell line comprising the step is provided: PCR amplification of the calmodulin gene or fragment thereof.

The PCR amplification of a fragment of the calmodulin gene allows the identification and / or differentiation of cell lines since the same genes of different cell lines differ in the length of introns. By

therefore, primer sets that are designed to amplify one or more of the introns found in the calmodulin gene can be used to identify and / or differentiate cell lines. In accordance with the foregoing, in one aspect of the disclosure, a method of identifying the cell line is provided comprising the step of: PCR amplification of one or more of the calmodulin introns or a fragment thereof. In a further aspect, a method of identifying the cell line is provided comprising the step of: PCR amplification of intron between exons 3 and 4 of the calmodulin gene.

In one aspect, a method is provided in which a fragment of the calmodulin gene is amplified by PCR and in a further aspect, a method is provided in which the amplified fragment comprises the sequence between exon 3 and exon 4 of the calmodulin gene.

In a further aspect, a method is provided in which a fragment of the calmodulin gene is amplified using at least one of the primers selected from SEQ ID No. 1 and SEQ ID No. 2 or a functional derivative thereof.

The Axl receptor tyrosine kinase gene has conserved exons and introns that vary between different cell lines. In accordance with the foregoing, in one aspect there is provided a method of identifying the cell line comprising the step: PCR amplification of an Axl receptor tyrosine kinase gene or fragment thereof. PCR amplification of a fragment of the Axi receptor tyrosine kinase gene allows differentiation between cell lines, since different cell lines differ in intron length. Therefore, sets of primers that are designed to amplify one or more of the introns found in the AxI receptor tyrosine kinase genes can be used to differentiate between different cell lines; thus the amplification of an intron that varies in length between the different cell lines can be used in the disclosure procedures. In accordance with the foregoing, in one aspect there is provided a method of identifying the cell line comprising the step: PCR amplification of one or more introns of the Axl receptor tyrosine kinase gene or fragment thereof. In a further aspect, a method of identifying the cell line is provided which comprises the step of: PCR amplification of the intron between exons 18 and 19 of the AxI receptor tyrosine kinase gene. In one aspect, a method is provided by which a fragment of the Axl receptor tyrosine kinase gene is amplified by PCR. In a further aspect, a method is provided in which the amplified fragment comprises the sequence between exon 18 and exon 19 of the Axl receptor tyrosine kinase gene. Suitably, the method in which a fragment of the Axl receptor tyrosine kinase gene is amplified utilizes at least one of the primers selected from SEQ ID No. 3 and SEQ ID No. 4 or a functional derivative thereof.

The type of cell can be determined by the presence or absence of the gene, that is, the particular cell line can be differentiated from another through the presence or absence of a particular gene or fragment thereof.

By "presence" of a gene it is meant that all or part of the specified gene is detectable by procedures well known to those skilled in the art.

For example, an insect cell line can be differentiated from a mammalian cell line through the detection of a gene only present in insect cells.

In accordance with the foregoing, in one aspect a method of identifying the cell line is provided which comprises the stage in which the type of cell line is determined by the presence and / or absence of an atacin gene or fragment thereof.

In accordance with the foregoing, in one aspect there is provided a method of identifying the cell line comprising the step: PCR amplification of the pre-proatacin gene (PPATT) or fragment thereof. In one aspect, a method of identifying the cell line is provided comprising the step: PCR amplification of the pre-proatacin A gene (PPATTA) or fragment thereof. In one aspect, a method is provided in which a fragment of the atacin gene is amplified by PCR. In accordance with the foregoing, a method in which a fragment of the PPATT gene is amplified using at least one of the primers comprising the sequences selected from SEQ ID No. 5 and SEQ ID No. 6 or a functional derivative thereof.

However, it is clear to the person skilled in the art that the presence and or absence of a gene can be determined by a series of techniques, including PCR amplification of the gene or part thereof. Other techniques by which the presence of genes can be evaluated include, but is not limited to, DNA hybridization, in particular in situ DNA hybridization. As used herein, the terms "hybridization" or "specific hybridization" means that the primer or probe forms a duplex (double stranded nucleotide sequence) with part of a target or with the entire region under experimental conditions. used, and that under such conditions, the primer or probe does not form a duplex with other regions of the nucleotide sequence present in the sample to be analyzed. It should be understood that the primers and probes of the present disclosure are designed for specific hybridization to specific genes and, therefore, may be completely within said region or may, to a large extent, overlap with said region (i.e., form a duplex with nucleotides outside, as well as within that region). In accordance with the above a procedure is provided in the

that primers as described herein can be used during a PCR reaction or DNA hybridization.

Polymerase chain reaction (PCR) amplification can be performed from the isolated and / or purified genomic nucleic acid from a cell culture. Covered in the disclosure are PCR reactions carried out in a cell suspension, crude genomic preparation, isolated or purified, using genomic DNA preparation techniques well known to those skilled in the art. Genomic preparations encompassed by the present disclosure can be isolated or substantially purified. By "isolated" or "substantially purified" it is intended that the nucleic acid molecules be substantially or essentially free of components normally found in association with the nucleic acid in its natural state. Such components include other cellular materials and culture media, for example.

Procedures for designing PCR primers are generally known in the art and are described in Sambrook and Russel, Molecular Cloning: A Laboratory Manual (Cold Harbor Laboratory Press). Known PCR procedures include, but are not limited to, procedures using paired primers, nested primers, individual specific primers, degenerate primers, gene specific primers, vector specific primers, partially matching primers, and the like.

With PCR, it is possible to amplify a single copy of a specific target sequence in the genomic DNA to a level detectable by several different methodologies (eg, without limitations, hybridization with a labeled probe; incorporation of biotinylated primers; followed by conjugate detection of avidin-enzyme; incorporation of 32P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment, incorporation of a fluorochrome such as ethidium bromide or other commercial compounds). In addition to genomic DNA, any nucleotide sequence can be amplified with the appropriate set of primer molecules. Specifically, the amplified segments created by the PCR procedure itself are, themselves, efficient molds for subsequent PCR amplifications.

PCR amplification requires "PCR reagents" or "PCR materials", which are defined herein as all reagents necessary to carry out the amplification, except for polymerase, primers and template. PCR reagents typically include nucleic acid precursors (dCTP, dTTP, etc.), and buffer.

The term "primer" is used herein to refer to any single chain oligonucleotide sequence that can be used as a primer in, for example, PCR technology. Therefore, a "primer" refers to a single chain oligonucleotide sequence that can act as a starting point for the synthesis of a primer extension product that is complementary to the nucleic acid strand to be copied. The design (specific length and sequence) of the primer will depend on the nature of the DNA and / or RNA targets and the conditions under which the primer is used (such as temperature and ionic strength).

Primers that may consist of the nucleotide sequences shown in SEQ ID NOS 1 to 6, or may be 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 15, 20, 25, 30, 35, 40, 45, 50, 75 or 100 or more bases that comprise or fall within the sequences of SEQ ID NO: 1 to 6, provided they are suitable for specific DNA binding of the target loci, under stringent conditions. When necessary, slight modifications of the primers or probes may be carried out in terms of length or sequence to maintain the specificity and sensitivity required in the given circumstances and, therefore, 1, 2, 3, 4, 5, 6 or more nucleotides may be substituted. The probes and / or primers listed herein may extend in 1, 2, 3, 4 or 5 nucleotides, for example, in any direction.

For the avoidance of doubt, the designation of a letter other than A, T, G and C means the following:

Single Letter Code

B = C or G or T

D = A or G or T

H = A or C or T

K = G or T

M = A or C

N = A or C or G or T

R = A or G

S = C or G

V = A or C or G

W = A or T

Y = C or T

The term "functional derivative" is used herein to refer to a primer comprising a sequence that is at least 95% identical to a primer as defined herein on the length of the primer, suitably greater than 95% identical such as 96%, 97%, 98%, 99% and, more preferably, has an identity of 100% over its length. Functional derivatives may have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base deletions at the 5 'or 3' end or both.

As used herein, the term "stringent conditions" means any hybridization condition that allows primers to bind to a specific nucleotide sequence, and not to any other locus in the cell genome. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A non-limiting example of stringent hybridization conditions is 6X hybridization of sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by one or more washes in 0.1 x SSC, 0.2% SDS at about 68 ºC. An alternative example of stringent hybridization conditions are hybridization in 6 X SSC at approximately 45 ° C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65 ° C (i.e., one or more washes at 50 ° C, 55 ° C, 60 ° C or 65 ° C).

The disclosure procedures can be adapted according to the cell lines that have to be differentiated or determined. For example, some cell lines can be differentiated by PCR amplification of a calmodulin-only intron. However, some cell lines may result in an amplicon that is the same size or as another cell line and, therefore, additional measures are required to identify the cell line.

In accordance with the foregoing, in one aspect there is provided a method of identifying the cell line comprising the steps: PCR amplification of the calmodulin gene or fragment thereof.

In a further aspect, a method of identifying the cell line is provided which comprises the steps of: PCR amplification of the atacin gene or fragment thereof.

In a further aspect, a cell line identification procedure is provided comprising the steps of: PCR amplification of the Axl receptor tyrosine kinase gene or fragment thereof.

In a further aspect, a cell line identification procedure is provided comprising the following steps: (a) PCR amplification of the calmodulin gene or fragment thereof; and (b) PCR amplification of an atacin gene or fragment thereof.

In a further aspect, a cell line identification procedure is provided comprising the following steps: (a) PCR amplification of the calmodulin gene or fragment thereof; and (b) PCR amplification of the Axl receptor tyrosine kinase gene or fragment thereof.

In a further embodiment, a cell line identification procedure is provided comprising the following steps: (a) PCR amplification of an atacin gene or fragment thereof; and (b) PCR amplification of an Axl receptor tyrosine kinase gene or fragment thereof.

In one aspect a method of identifying the cell line is provided comprising the following steps: (a) PCR amplification of the calmodulin gene or fragment thereof; (b) PCR amplification of an atacin gene or fragment thereof; and (c) PCR amplification of the Axl receptor tyrosine kinase gene

or fragment thereof.

In a particular aspect, PCR reactions can be carried out in a single PCR reaction. In accordance with the foregoing, one aspect provides a method in which PCR amplification reactions are multiplexed. As used herein, the term "multiplexing" means that any number of PCR reactions is performed in a single reaction tube, that is, the content of a single reaction tube comprises more than 1 set of primers. The primers of the disclosure are designed so that a single PCR reaction can be performed using the specified reaction conditions that allow amplifications of each gene or fragment thereof used in the cell identification procedure (calmodulin, receptor tyrosine kinase Axl and atacina) without prejudice to the amplification of another gene. Multiplexing allows a faster and easier method of molecular identification of the cell line as described herein.

The methods of the disclosure are suitable for the identification of several cell lines comprising a calmodulin gene, Axl receptor tyrosine kinase or atacin. In one aspect, a

procedure for the identification of cell lines used in the pharmaceutical industry, particularly in the production of vaccines. In one aspect, a procedure is provided for the identification of the MRC-5, Vero, Hi-5, CHO, FRHL2, CEF and MDCK cell lines. These cell lines are well known to those skilled in the art, however, to summarize:

Cellphone line

Organism

CHO

Cricetulus griseus Chinese hamster

MRC-5

Homo sapiens being human

Canis familiaris

MDCK

Dog

Vero

Cercopithecus aethiops African green monkey

Macaca mulatta

FRHL2

Rhesus macaque

Chicken

Gallus gallus

Hi-5

Trichoplusia or Lepidopteran Insects

In a further aspect a kit is provided for the identification of the cell line comprising primers complementary to any one of: the calmodulin gene; an Axl receptor tyrosine kinase gene; or an attack gene; or any combination thereof. In a further aspect, the kit comprises at least one primer

10 comprising or consisting of the following sequences or functional derivatives thereof: SEQ ID NO: 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6 or a functional derivative thereof.

In one aspect, the use of a calmodulin gene, a tyrosine kinase gene and / or the atacin gene or any combination thereof in the identification of the cell line is provided.

Examples

15 1.1 Genomic DNA extraction and PCR amplification

Genomic DNA was extracted from CHO, MRC-5, MDCK, VERO, FRHL2, Hi-5 and Gallus [CFO] cells using the High Pure PCR template Preparation Kit (Roche). The PCR was then performed under the following reaction conditions using the primers Calm ex 3F (SEQ ID No. 1 Calm ex 4R (SEQ ID No. 2), Axl ex 18F (SEQ ID No. 3), Axl 19R (SEQ ID No. 4), ppatta F (SEQ ID No. 5) and ppatta R (SEQ ID No. 6)

Mixture

Cycles

Volume (μl)

Ingredients Temperature Weather Cycles

up to 25

H2OPCR 95 ° C 4 min

2.5

Tp PCR10x (+ MgC12) 95 ° C 30 s X35

one

10 mM dNTP 60 ° C 30 s

0.2

Axl primer exl 8F 72 ° C 1 min

0.2

Axl primer exl 9R 72 ° C 7 min

0.1

Calm ex 3F primer

0.1

Calm ex 4R primer

0.05

Ppatta primer F

0.05

Ppatta R primer

0.3

Ex Taq Takara

one

DNA (100 ng)

2.1 Determination of the size of PCR products by capillary electrophoresis

The following example demonstrates that the difference in intron size between exons 3 and 4 of calmodulin and 18 and 19 of Axl, can unequivocally identify the 7 different cell lines from which genomic DNA 5 was extracted (see 1.1 ).

In order to allow the detection of DNA products after capillary electrophoresis, the reverse primers used during the PCR amplification were labeled with different fluorescent dyes; the reverse primer for PPTTA was marked with NED, the reverse primer for calmodulin was labeled with Hex and the reverse primer for Axl was labeled with 6-FAM. The resulting labeled DNA fragments were run in parallel with a marker of

10 size, which allows the analysis of the PCR product with a size resolution of approximately 1 bp for fragments of less than 500 bp.

Genomic DNA was amplified using fluorescent inverse primers at a hybridization temperature of 60 ° C (according to the PCR conditions described above). Capillary electrophoresis was performed with the Genetic Analyzer instrument (ABl3730) using 50 cm capillaries (ladder ROX-1200, ABI).

15 The sizes of the amplified products were compared with the sizes expected in Table 1. Capillary electrophoresis profiles are presented in Figures 1 to 3.

Table 1: Comparison of the sizes of the PCR products determined by capillary electrophoresis (observed from the cells) with the sizes deduced from the published sequences (expected from the organism).

PPATTA cells

CALM3 / 4 AXL18 / 19

Size (bp) Observed cells Expected organism

Observed cells Expected organism Observed cells Expected organism

MRC-5 NA NA

316 320 1001 1010

VERO NA NA

273 ND 1027 + 1031 ND

CHO NA NA

274 ND 654 ND

FRHL2 NA NA

274 278 1007 1021

MDCK NA NA

277 + 279 283 355 + 577 583

Chicken NA NA

267 270 1350 1489

Hi-5 244 235

NA NA NA NA

NA: Not applicable ND: Not described

CALM3 / 4 PCR products (amplification of the calmodulin gene between exons 3 and 4)

The size of the amplified PCR products from the MRC-5, FRHL-2, MDCK and chicken cells was very close to the expected sizes. Two peaks were observed from MDCK cells (277 and 279 bp).

A 274bp fragment was amplified from CHO and FHRL-2 cells, near the 273 bp fragment amplified from VERO cells. Other fragments were different in at least 3 base pairs. The sizes of the VERO and CHO fragments were not known before performing the experiment.

PCR products AXL18 / 19 (amplification of the calmodulin gene between exons 18 and 19)

The AXL 18/19 PCR allowed the discrimination of the DNA of 6 vertebrates. The size of the products amplified from VERO cells (1,027 and 1,031 bp) and CHO (654 bp) was clearly different.

PPATTA PCR products (atacina)

The size of the DNA fragment amplified from T. DNA or using the PPATTA primers was estimated at 244 bp. This size coincided with the expected 235 bp.

Conclusions

The combined analysis of the PCR products of CALM3 / 4 and AXL18 / 19 produced size patterns that allow unambiguous identification of the 6 vertebrate cell lines.

3.1 Ability to detect a smaller species that is, pollution

The objective of the following example was to evaluate the ability of the previous PCR assay to detect a mixture of 2 different cell types in which the main species represent 90%, and the smaller species represent 10% of the mixture.

Mixtures of 90/10% of different cell types were performed at the genomic DNA level considering the genome copy number (see Table 2).

The number of copies of the genome was calculated from the amount of DNA (measured by the Threshold system) and the genome sizes (http://www.genomesize.com).

Table 2: Determination of the number of copies of the genome

Cellphone line

Haploid genome size (qg) DNA concentration (ng / µl) Volume in 100 µl to obtain 9,000 copies / µl Volume in 100 µl to get 1000 copies / µl

MRC-5

3.5 83 37.8 4.2

Vero

3.5 58 54 6

Hi-5

0.5 96 5 0.5

CHO

3.5 69 Four. Five 5

FRHL2

3.5 126 25.2 2.8

MDCK

3.2 3. 4 85.5 9.5

Chicken

1.25 38 29.7 3.3

Approximately 27,000 copies of the main species and 3,000 copies of the minor species were mixed and amplified under the PCR conditions described above (see 1.1)

Results The results of the detection of the different mixtures are summarized in Table 3. Table 3: Detection of a minor species present in 10% in the cell line mixture

Line

MRC-5 VERO Hi-5 CHO FRHL-2 MDCK Chicken

mobile

Gen 10% 10% 10% 10% 10% 10% 10%

Calm

okay NA okay okay okay okay

MRC-5

90% Axl okay NA okay okay okay okay

PPATTA

NA okay NA NA NA NA

VERO

90% Calm okay NA 278/277 278/277 okay okay

Axl

NO NA okay okay okay okay

PPATTA

NA okay NA NA NA NA

Hi-5

90% Calm okay okay okay okay okay okay

Axl

okay okay okay okay okay okay

PPATTA

NA NA NA NA NA NA

CHO

90% Calm okay 277/278 NA 278/278 okay okay

Axl

okay okay NA okay okay okay

PPATTA

NA NA okay NA NA NA

FRHL-2

90% Calm okay 277/278 NA 278/278 NO okay

Axl

NO okay NA okay okay okay

PPATTA

NA NA okay NA NA NA

MDCK

90% Calm okay okay NA okay okay okay

Axl

okay okay NA okay okay okay

PPATTA

NA NA okay NA NA NA

Chicken

90% Calm okay okay NA okay okay okay

Axl

okay okay NA okay okay okay

PPATTA

NA NA okay NA NA NA

OK: 10% of the minor species were detected using the aforementioned PCR. When the species cannot be distinguished because the fragment size is identical, or very close, the size of both products is indicated in the table.

5 NO: 10% of minor species were not detected using the aforementioned PCR NA: Not applicable For some mixtures, a single PCR amplification did not allow the detection of minor species:

 CHO / VERO / FRHL2 mixtures could not be detected using CALM3 / PCR because the products showed identical or similar sizes 10  10% of MDCK in FRHL-2 could not be detected with the CALM3 / 4 assay

 10% of MRC5 in FRHL-2 and in VERO could not be detected with the AXL18 / 19 assay. Therefore, it can be seen that the combined analysis of the PCR products of CALM3 / 4, AXL18 / 19 and PPATTA allowed the detection of a 10% DNA contamination between the 7 different cell lines and that, in some cases, only 1 or 2 gene analysis was required to detect the contamination.

15 Sequence listing

<110> Cassart, Jean-Pol Lienard, Patricia

<120> New procedure

<130> VB63206P

<160> 9 20 <170> FastSEQ for Windows version 4.0

<210> 1

<211> 27

<212> DNA

<213> Artificial sequence 25 <220>

<223> oligonucleotide

<400> 1 caaagaagcy ttttcactat ttgacaa 27

<210> 2 30 <211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

5

<400> 2

tcatttttct tgccatcatt gtcaga

26

<210> 3

<211> 20

<212> DNA

10

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 3

ggrgactact accgycaggg

twenty

fifteen

<210> 4

<211> 22

<212> DNA

<213> Artificial sequence

<220>

twenty

<223> oligonucleotide

<400> 4

caatctccca catbgtcacc cc

22

<210> 5

<211> 25

25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 5

30

ctattgtcga ccatacctct accgt 25

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

35

<220>

<223> oligonucleotide

<400> 6

cgtggccgtt gctggataag 20

<210> 7

<211> 149

<212> PRT

<213> H. sapiens

<400> 7

<210> 8

<211> 894

<212> PRT

<213> H. sapien

<400> 8

<210> 9

<211> 192

<212> PRT

<213> Trichoplusia

<400> 9

Claims (2)

1. A cell line identification procedure comprising the following steps: (a) calmodulin gene analysis, in which a fragment of the calmodulin gene is amplified by PCR and wherein the amplified fragment comprises the sequence between exon 3 and exon 4 of the calmodulin gene; Y (b) analysis of the Axl receptor tyrosine kinase gene, in which a fragment of the Axl receptor tyrosine kinase gene is amplified by PCR, and in which the amplified fragment comprises the sequence between exon 18 and exon 19 of the gene of the receptor tyrosine kinase Axl; and in which the cell line is identified by the size of an intron in said genes. The method of claim 1, wherein the calmodulin gene is a nucleic acid sequence that encodes a polypeptide of SEQ ID No. 7 or encodes an amino acid sequence that has a sequence identity of 70%, 75 %, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO. 7. 3. The method of claim 1 or 2, wherein the Axl receptor tyrosine kinase gene is a nucleic acid sequence encoding a polypeptide of SEQ ID No. 8 or polypeptides with 15 amino acid sequences having a sequence identity of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98% or 99% with SEQ ID NO. 8.